ABC transporter

ABSTRACT

The invention provides ABC transporter polypeptides and polynucleotides encoding ABC transporter polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ABC transporter polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/946,348, filed Oct. 7,1997, now abandoned.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the ABC transporterfamily, as well as their variants, hereinafter referred to as “ABCtransporter,” “ABC transporter polynucleotide(s),” and “ABC transporterpolypeptide(s)” as the case may be.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and gencproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce tvo types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Staphylococciis aureus infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Staphylococcus aureus strains which are resistant tosome or all of the standard antibiotics. This phenomenon has created anunmet medical need and demand for new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the ABC transporter embodiments of the invention, that have a presentbenefit of, among other things, being useful to screen compounds foranitimicrobial activity. Such factors are also useful to determine theirrole in pathogenesis of infection, dysfunction and disease. There isalso a need for identification and characterization of such factors andtheir antagonists and agonists to find ways to prevent, ameliorate orcorrect such infection, dysflunction and disease.

SUMMARY OF THE INVENTION

The present invention relates to ABC transporter, in particular ABCtransporter polypeptides and ABC transporter polynucleotides,recombinant materials and methods for their production. In anotheraspect, the invention relates to methods for using such polypeptides andpolynucleotides, including treatment of microbial diseases, amongstothers. In a further aspect, the invention relates to methods foridentifying agonists and antagonists using the materials provided by theinvention, and for treating microbial infections and conditionsassociated with such infections with the identified agonist orantagonist compounds. In a still further aspect, the invention relatesto diagnostic assays for detecting diseases associated with microbialinfections and conditions associated with such infections, such asassays for detecting ABC transporter expression or activity.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to ABC transporter polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a ABCtransporter of Staphylococcus aureus, which is related by amino acidsequence homology to ABC transporter, ATP-binding protein [Arheoglobusfulgidus] AE001033 polypeptide. The invention relates especially to ABCtransporter having the nucleotide and amino acid sequences set out inTable 1 as SEQ ID NO:1 and SEQ ID NO:2 respectively. Note that sequencesrecited in the Sequence Listing below as “DNA” represent anexemplification of the invention, since those of ordinary skill willrecognize that such sequences can be usefully employed inpolynucleotides in general, including ribopolynucleotides.

TABLE 1 ABC transporter Polynucleotide and Polypeptide Sequences (A)Staphylococcus aureus ABC transporter polynucleotide sequence [SEQ IDNO:1]. 5′-TGTGGATCCTATCGAAGCAATTGGAGGTGCAGAATAATGGCATTAGTCGTTAAAGATATCGTCAAAAATTTCGGAGAAGGTTTGTCTGAAACAAAAGTTTTAAAAGGTATTAATTTTGAAGTGGAACAAGGGGAATTTGTCATTTTAAATGGTGCCTCTGGTTCTGGGAAAACAACATTGCTAACGATATTAGGCGGATTGTTAAGTCAAACGAGTGGTACAGTGCTTTACAATGATGCACCATTGTTTGATAAACAGCATCCTCCTAGTGATTTGCGATTGGAAGATATTGGTATTATTATTCAATCTTCACATATAGTTCCTTATTTAAAAGTGATAGAGCAATTGACACTAGTAGGCCAAGAAGCGGGAATGACCAAACAACAAAGTTCAACAAGAGCAATACAACTTTTGAAAAATATTGGGTTAGAAGATCGCTTGAATGTATATCCGCATCAGTTATCTGGCGGTGAAAAGCAACGTGTTGCGATTATGAGAGCATTTATGAATAATCCGAAAATCATTTTAGCAGATGAGCCCACAGCAAGCTTAGATGCCGATAGAGCAAAAAAAGTTGTTGAGATGATACGTCAACAAATTAAAGAACAACAAATGATTGGTATTATGATTACACACGATCGAAGATTATTTGAATATGCAGATAGAGTGATAGAATTAGAAGATGGCAAAATAACTGATTAG-3′ (B)Staphylococcus aureus ABC transporter polypeptide sequence deduced froma polynucleotide sequence in this table [SEQ ID NO:2]. NH₂-CGSYRSNWRCRIMALVVKDIVKNFGEGLSETKVLKGINFEVEQGEFVILNGASGSGKTTLLTILGGLLSQTSGTVLYNDAPLFDKQHPPSDLRLEDIGIIIQSSHIVPYLKVIEQLTLVGQEAGMTKQQSSTRAIQLLKNIGLEDRLNVYPHQLSGGEKQRVAIMRAFMNNPKIILADEPTASLDADRAKKVVEMIRQQIKEQQMIGIMITHDRRLFEYADRVIELEDGKITD-COOH

Deposited Materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains a full length ABC transporter gene. Thesequence of the polynucleotides contained in the deposited strain, aswell as the amino acid sequence of any polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A liceinse may be required to make, useor sell the deposited strain, and compounds derived therefrom, and nosuch license is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStaphylococcus aureus WCUH 29 strain, which polypeptide is contained inthe deposited strain. Further provided by the invention are ABCtransporter polynucleotide sequences in the deposited strain, such asDNA and RNA, and amino acid sequences encoded thereby. Also provided bythe invention are ABC transporter polypeptide and polynucleotidesequences isolated from the deposited strain.

Polypeptides

ABC transporter polypeptide of the invention is substantiallyphylogenetically related to other proteins of the ABC transporterfamily.

In one aspect of the invention there are provided polypeptides ofStaphylococcus aureus referred to herein as “ABC transporter” and “ABCtransporter polypeptides” as well as biologically, diagnostically,prophylactically, clinically or therapeutically useful variants thereof,and compositions comprising the same.

Among the particularly preferred embodiments of the invention arevariants of ABC transporter polypeptide encoded by naturally occurringalleles of the ABC transporter gene. The present invention furtherprovides for an isolated polypeptide which: (a) comprises or consists ofan amino acid sequence which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, most preferably at least 97-99% orexact identity, to that of SEQ ID NO:2 over the entire length of SEQ IDNO:2; (b) a polypeptide encoded by an isolated polynucleotide comprisingor consisting of a polynucleotide sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO:1 over theentire length of SEQ ID NO:1; (c) a polypeptide encoded by an isolatedpolynucleotide comprising or consisting of a polynucleotide sequenceencoding a polypeptide which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%or exact identity, to the amino acid sequence of SEQ ID NO:2, over theentire length of SEQ ID NO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofABC transporter, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NO:1] or the relevant portion, preferablyat least 80% identity to a polypeptide of Table 1 [SEQ ID NO:2] and morepreferably at least 90% identity to a polypeptide of Table 1 [SEQ IDNO:2] and still more preferably at least 95% identity to a polypeptideof Table 1 [SEQ ID NO:2] and also include portions of such polypeptideswith such portion of the polypeptide generally containing at least 30amino acids and more preferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxyltenninus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where in and/or n is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStaphylococcus aureus, however, it may preferably be obtained from otherorganisms of the same taxonomic genus. A polypeptide of the inventionmay also be obtained, for example, from organisms of the same taxonomicfamily or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypcptide of the invention. As with ABC transporter polypeptides,fragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Staphylococcus aureus, are also preferred. Furtherpreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Also preferred are biologically active fragments which are thosefragments that mediate activities of ABC transporter, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate. or maintain cause Disease in anindividual, particularly a human.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

In addition to the standard single and triple letter representations foramino acids, the term “X” or “Xaa” may also be used in describingcertain polypeptides of the invention. “X” and “Xaa” mean that any ofthe twenty naturally occurring amino acids may appear at such adesignated position in the polypeptide sequence.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodeABC transporter polypeptidcs, particularly polynucleotides that encodethe polypeptide herein designated ABC transporter.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding ABC transporter polypeptidescomprising a sequence set out in Table 1 [SEQ ID NO:1] which includes afull length gene, or a variant thereof The Applicants believe that thisfull length gene is essential to the growth and/or survival of anorganism which possesses it, such as Staphylococcus aureus.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing ABC transporter polypeptidesand polynucleotides, particularly Staphylococcus aureus ABC transporterpolypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

Another aspect of the invention relates to isolated polylnucleotides,including at least one full length gene, that encodes a ABC transporterpolypeptide having a deduced amino acid sequence of Table 1 [SEQ IDNO:2] and polynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is aABC transporter polypeptide from Staphylococcus aureus comprising orconsisting of an amino acid sequence of Table 1 [SEQ ID NO:2], or avariant thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding ABC transporter polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a polynucleotide sequence given in Table 1 [SEQ IDNO:1], typically a library of clones of chromosomal DNA ofStaphylococcus aureus WCUH 29 in E. coli or some other suitable host isprobed with a radiolabeled oligonucleotide, preferably a 17-mer orlonger, derived from a partial sequence. Clones carrying DNA identicalto that of the probe can then be distinguished using stringenthybridization conditions. By sequencing the individual clones thusidentified by hybridization with sequencing primers designed from theoriginal polypeptide or polynticleotide sequence it is then possible toextend the polynucleotide sequence in both directions to determine afull length gene sequence. Conveniently, such sequencing is performed,for example, using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Direct genomic DNAsequencing may also be performed to obtain a full length gene sequence.Illustrative of the invention, each polynucleotide set out in Table 1[SEQ ID NO:11] was discovered in a DNA library derived fromStaphylococcus aureus WCUH 29.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon which begins at nucleotide number 700 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynucleotidesequence which has at least 70% identity, preferably at least 80%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, even more preferably at least 97-99% or exactidentity to SEQ ID NO:1 over the entire length of SEQ ID NO:1, or theentire length of that portion of SEQ ID NO:1 which encodes SEQ ID NO:2;(b) a polyniucleotide sequence encoding a polypeptide which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or 100% exact, to the amino acid sequence ofSEQ ID NO:2, over the entire length of SEQ ID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Staphylococciusaureus, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof, andisolating a full-length gene and/or genomic clones containing saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al, Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al, Cell 37: 767 (1984), both ofwhich may be useful in purifyng polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotidc 1 to the nucleotide immediatelyupstream of or including nucleotide 700 set forth in SEQ ID NO:1 ofTable 1, both of wlich encode the ABC transporter polypeptide.

The invention also includes a polynuclcotide consisting of or comprisinga polynucleotide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3′ end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof In the polynucleotide formula above, R₂ isoriented so that its 5′ end nucleic acid residue is at the left, boundto R₁, and its 3′ end nucleic acid residue is at the right, bound to R₃.Any stretch of nucleic acid residues denoted by either R₁ and/or R₂,where m and/or n is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, which can bea double-stranded polynucleotide wherein the formula shows a firststrand to which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Staphylococcus aureus, however, it may preferably be obtained fromother organisms of the samne taxonomic genus. A polynucleotide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus ABCtransporter having an amino acid sequence set out in Table 1 [SEQ IDNO:2]. The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, polynucleotides interrupted by integrated phage, an integratedinsertion sequence, an integrated vector sequence, an integratedtransposon sequence, or due to RNA editing or genomic DNAreorganization) together with additional regions, that also may containcoding and/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments of apoly-nucleotides of the invention may be used, for example, tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingABC transporter variants, that have the amino acid sequence of ABCtransporter polypeptide of Table 1 [SEQ ID NO:2] in which several, afew, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, modified, deleted and/or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, that do not alter the properties and activities of ABCtransporter polypeptide.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding ABC transporter polypeptide having an amino acid sequence setout in Table 1 [SEQ ID NO:2], and polynucleotides that are complementaryto such polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding ABC transporterpolypeptide and polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical over their entire length to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to ABC transporter polynucleotide sequences, suchas those polynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof, and isolating said polynucleotide sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, CDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding ABC transporter and toisolate CDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to the ABC transportergene. Such probes generally will comprise at least 15 nucleotideresidues or base pairs. Preferably, such probes will have at least 30liucleotide residues or base pairs and may have at least 50 nucleotideresidues or base pairs. Particularly preferred probes will have at least20 nucleotide residues or base pairs and will have Ice than 30nucleotide residues or base pairs.

A coding region of a ABC transporter gene may be isolated by screeningusing a DNA sequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genoilc DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, el al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the teclmique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to almeal within theamplified product (typically an adaptor specific primer that amiealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as fiiurer discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is the mature protein plus additional amino or carboxyl-terminalamino acids, or aio acids interior to the mature polypeptide (when themature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from the mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature fonn of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polinucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant teclmiques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systemswhich comprise a polynucleotide or polynucleotides of the presentinvention, to host cells which are genetically engineered with suchexpression systeimns, and to the production of polypeptides of theinvention by recombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, el al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, el al., MOLECULARCLONING: A LIABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated trmnsfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci E. coli,streptoinyces, cyanobacteria, Bacillus subtilis, and Staphylococcusaureus; fingal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagernids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukarotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-knoun methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of ABC transporterpolynucleotides and polypeptides of the invention for use as diagnosticreagents. Detection of ABC transporter polynucleotides and/orpolypeptides in a eukaryote, particularly a mammal, and especially ahuman, will provide a diagnostic method for diagnosis of disease,staging of disease or response of an infectious organism to drugs.Eukaryotes, particularly mammals, and especially humans, particularlythose infected or suspected to be infected with an organism comprisingthe ABC transporter gene or protein, may be detected at the nucleic acidor amino acid level by a variety of weell knows techniques as well as bymethods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, imnay be used directly for detectionor may be amplified enzymatically by using PCR or any otheramplification technique prior to analysis. RNA, particularly mRNA, cDNAand genormic DNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled ABC transporter polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexcsby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the clectrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence changes at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V₁and S₁ protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Nati. Acad Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingABC transporter nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonolmic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit which comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, which isassociated ith a disease or pathogenicity will provide a diagnostic toolthat can add to, or define, a diagnosis of a disease, a prognosis of acourse of disease, a determination of a stage of disease, or asusceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof teclmques, such as those described elsewhere herein.

The nucleotide sequences of the present invention are also valuable fororganism chromosome identification. The sequence is specificallytargeted to, and can hybridize with, a particular location on anorganism's chromosome, particularly to a Staphylococcus aureuschromosome. The mapping of relevant sequences to chromosomes accordingto the present invention may be an important step in correlating thosesequences with pathogenic potential and/or an ecological niche of anorganism and/or drug resistance of an organism, as -well as theessentiality of the gene to the organism. Once a sequence has beenmapped to a precise chrornosomal location, the physical position of thesequence on the chromosome can be correlated Wvith genetic map data.Such data may be found on-line in a sequence database. The relationshipbetween genes and diseases that have been mapped to the sameclhromosomal region are then identified through knoan genetic methods,for example, through linkage analysis (coinheritance of physicallyadjacent genes) or mating studies, such as by conjugation.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynuclcotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, CDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding ABC transporter polypeptidc can be used toidentify and analyze mutations. Examples of representative prirners areshow below in Table 2.

TABLE 2 Primers for amplification of ABC transporter polynucleotides SEQID NO PRIMER SEQUENCE 3 5′-ACTAGTAGGCCAAGAAGCGGG-3′ 45′-TGCTCTCATAATCGCAACACGT-3′

The invention also includes primers of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

wherein, at the 5′ end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, and at the 3′ end of the molecule, Y ishydrogen, a metal or a modified nucleotide residue, R₁ and R₃ are anynucleic acid residue or modified nucleotide residue, m is an integerbetween 1 and 20 or zero , n is an integer between 1 and 20 or zero, andR₂ is a primer sequence of the invention, particularly a primer sequenceselected from Table 2. In the polynucleotide formula above R₂ isoriented so that its 5′ end nucleotide residue is at the left, bound toR₁, and its 3′ end nucleotide residue is at the right, bound to R₃. Anystretch of nuclcic acid residues denoted by either R group, where mand/or n is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer being complementary to a regionof a polynucleotide of Table 1. In a preferred embodiment m and/or n isan integer between 1 and 10.

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifig ABC transporter DNA and/or RNAisolated from a sample derived from an individual, such as a bodilymaterial. The primers may be used to amplify a polynucleotide isolatedfrom an infected individual, such that the polyntucleotide may then besubject to various techniques for elucidation of the polynucleotidesequence. In this way, mutations in the polynucleotide sequence may bedetected and used to diagnose and/or prognose the infection or its stageor course, or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStaphylococcus aureus, comprising determining from a sample derived froman individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a ABC transporter polynucleotidecan be measured using any on of the methods well known in the art forthe quantitation of polynucleotides, such as, for example,amplification, PCR, RT-PCR, RNase protection, Northern blotting,spectrometry and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of ABC transporter polypeptide compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay teclniques that can be used to determinelevels of a ABC transporter polypeptide, in a sample derived from ahost, such as a bodily material, are well-known to those of skill in theart. Such assay methods include radioimmunoassays, competitive-bindingassays, Western Blot analysis, antibody sandwich assays, antibodydetection and ELISA assays.

Differential Expression

The polynucleotides and polynucleotides of the invention may be used asreagents for differential screening methods. There are many differentialscreening and differential display methods known in the art in which thepolynucleotides and polypeptides of the invention may be used. Forexample, the differential display technique is described by Chuang etal., J Bacteriol. 175:2026-2036 (1993). This method identifies thosegenes which are expressed in an organism by identifying mRNA presentusing randomly-primed RT-PCR. By comparing pre-infection and postinfection profiles, genes up and down regulated during infection can beidentified and the RT-PCR product sequenced and matched to ORF“unknowns.”

In Vivo Expression Technology (IVET) is described by Camilli et al,Proc. Nat'l. Acad. Sci. USA. 91:2634-2638 (1994). IVET identifies genesup-regulated during infection when compared to laboratory cultivation,implying an important role in infection. ORFs identified by thistechnique are implied to have a significant role in infectionestablishment and/or maintenance. In this technique random chromosomalfragments of target organism are cloned upstream of a promoter-lessrecombinase gene in a plasmid vector. This construct is introduced intothe target organism which carries an antibiotic resistance gene flankedby resolvase sites. Growth in the presence of the antibiotic removesfrom the population those fragments cloned into the plasmid vectorcapable of supporting transcription of the recombinase gene andtherefore have caused loss of antibiotic resistance. The resistant poolis introduced into a host and at various times after infection bacteriamay be recovered and assessed for the presence of antibiotic resistance.The chromosomal fragment carried by each antibiotic sensitive bacteriumshould carry a promoter or portion of a gene normally upregulated duringinfection. Sequencing upstream of the recombinase gene allowsidentification of the up regulated gene.

RT-PCR may also be used to analyze gene expression patterns. For RT PCRusing the polynucleotides of the invention, messenger RNA is isolatedfrom bacterial infected tissue, e.g., 48 hour murine lung infections,and the amount of each mRNA species assessed by reverse transcription ofthe RNA sample primed with random hexanucleotides followed by PCR withgene specific primer pairs. The determination of the presence and amountof a particular mRNA species by quantification of the resultant PCRproduct provides information on the bacterial genes which aretranscribed in the infected tissue. Analysis of gene transcription canbe carried out at different times of infection to gain a detailedknowledge of gene regulation in bacterial pathogenesis allowing for aclearer understanding of which gene products represent targets forscreens for antibacterials. Because of the gene specific nature of thePCR primers employed it should be understood that the bacterial mRNApreparation need not be free of mammalian RNA. This allows theinvestigator to carry out a simple and quick RNA preparation frominfected tissue to obtain bacterial mRNA species which are very shortlived in the bacterium (in the order of 2 minute halflives). Optimallythe bacterial mRNA is prepared from infected murine lung tissue bymechanical disruption in the presence of TRIzole (GIBCO-BRL) for veryshort periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of Staphylococcus aureus 16Sribosomal RNA as detected by probing Northerns with a suitably labeledsequence specific oligonucleotide probe. Typically a 5′ dye labeledprimer is used in each PCR primer pair in a PCR reaction which isterminated optimally between 8 and 25 cycles. The PCR products areseparated on 6% polyacrylamide gels with detection and quantificationusing GeneScanner (manufactured by ABI).

Gridding and Polynucleotide Subtraction

Methods have been described for obtaining information about geneexpression and identity using so called “high density DNA arrays” orgrids. See, e.g., M. Chee et al., Science, 274:610-614 (1996) and otherreferences cited therein. Such gridding assays have been employed toidentify certain novel gene sequences, referred to as Expressed SequenceTags (EST) (Adams et a., Science, 252:1651-1656 (1991)). A variety oftechniques have also been described for identifying particular genesequences on the basis of their gene products. For example, seeInternational Patent Application No. W091/07087, published May 30, 1991.In addition, methods have been described for the amplification ofdesired sequences. For example, see International Patent Application No.W091/17271, published Nov. 14, 1991.

The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probes obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularlyStaphylococcus aureus, and may be useful in diagnosing and/or prognosingdisease or a course of disease. A grid comprising a number of variantsof the polynucleotidc sequence of SEQ ID NO:1 are preferred. Alsopreferred is a comprising a number of variants of a polynucleotidesequence encoding the polypeptide sequence of SEQ ID NO:2.

Antibodies

The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as uni-unogens toproduce antibodies immuospecific for such polypeptides orpolynucleotides respectively.

In certain preferred embodiments of the invention there are providedantibodies against ABC transporter polypeptides or polynucleotides.

Antibodies generated against the polypeptides or polyniucleotides of theinvention can be obtained by administering the polypeptides and/orpolynucleotides of the invention, or epitope-bearing fragments of eitheror both, analogues of either or both, or cells expressing either orboth, to an ainimal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstcin, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4:72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-ABC transporter orfrom naive libraries (McCafferty, et al., (1990), Nature 348, 552-554;Marks, et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by, for example, chain shuffling(Clackson et al., (1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides or polynucleotides of the inventionto purifv the polypeptides or polynucleotides by, for example, affinitychromatography.

Thus, among others, antibodies against ABC transporter-polypeptide orABC transporter-polynucleotide may be employed to treat infections,particularly bacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

A polypeptide or polnucleotide of the invention, such as anantigenically or immunologically equivalent derivative or a fusionprotein of the polypeptide is used as an antigen to immunize a mouse orother animal such as a rat or chicken. The fusion protein may providestability to the polypeptide. The antigen may be associated, for exampleby conjugation, with an immunogenic carrier protein for example bovineserum albumin, keyhole limpet haemocyanin or tetanus toxoid.Alternatively, a multiple antigenic polypeptide comprising multiplecopies of the polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized,” where thecomplimentarily determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

In accordance with an aspect of the invention, there is provided the useof a polynucleotide of the invention for therapeutic or prophylacticpurposes, in particular genetic immunization. Among the particularlypreferred embodiments of the hivention are naturally occurring allelicvariants of ABC transporter polynucleotides and polypeptides encodedthereby.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet(1992) 1:363, Manthorpe et al., Hum. Gene Ther. (1983) 4:419), deliveryof DNA complexed with specific protein carriers (Wu et al., J Biol Chem.(1989) 264: 16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS USA, (1986) 83:9551), encapsulation of DNA invarious forms of liposomes (Kaneda et al., Science (1989) 243:375),particle bombardment (Tang et al., Nature (1992) 356:152, Eisenbraun etal., DNA Cell Biol (1993) 12:791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS USA (1984) 81: 5849).

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or flmctional mimetics. See, e.g.,Coligani et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases hereinbefore mentioned. It istherefore desirable to devise screening methods to identify compoundswhich stimulate or which inhibit the function of the polypeptide orpolynucleotide. Accordingly, in a further aspect, the present inventionprovides for a method of screening compounds to identify those whichstimulate or which inhibit the function of a polypeptide orpolynucleotide of the invention, as well as related polypeptides andpolynucleotides. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-frce preparations, chemical libraries, aid naturalproduct nixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of ABC transporter polypeptides andpolynucleotides; or may be structural or functional mfietics thereof(see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5(1991)).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotidc, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynuclcotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide or polynucleotide, as the case may be.Further, the screening methods may simply comprise the steps of mixing acandidate compound with a solution containing a polypeptide orpolynucleotide of the present invention, to form a mixture, measuringABC transporter polypeptide and/or polynucleotide activity in themixture, and comparing the ABC transporter polypeptide and/orpolynucleotide activity of the mixture to a standard. Fusion proteins,such as those made from Fc portion and ABC transporter polypeptide, ashereinbefore described, can also be used for high-throughput screeningassays to identify antagonists of the polypeptide of the presentinvention, as well as of phylogenetically and and/or functionallyrelated polypeptides (see D. Bennett el al., J Mol Recognition, 8:52-58(1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentswhich may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of ABCtransporter polypeptides or polynucleotides, particularly thosecompounds that are bacteristatic and/or bactericidal. The method ofscreening may involve high-throughput techniques. For example, to screenfor agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising ABC transporter polypeptide and alabeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be a ABCtransporter agonist or antagonist. The ability of the candidate moleculeto agonize or antagonize the ABC transporter polypeptide is reflected indecreased binding of the labeled ligand or decreased production ofproduct from such substrate. Molecules that bind gratuitously, i.e.,without inducing the effects of ABC transporter polypeptide are mostlikely to be good antagonists. Molecules that bind well and, as the casemay be, increase the rate of product production from substrate, increasesignal transduction, or increase chemical channel activity are agonists.Detection of the rate or level of, as the case may be, production ofproduct from substrate, signal transduction, or chemical channelactivity may be enhanced by using a reporter system. Reporter systemsthat may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in ABC transporter polynucleotide orpolypeptide activity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance,¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide which compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by ABC transporter polypeptide associatingwith another ABC transporter polypeptidc or other polypeptide, labeledto comprise a fluorescently-labeled molecule will have higherpolarization values than a fluorescently labeled monomeric protein. Itis preferred that this method be used to characterize small moleculesthat disrupt polypeptide complexes.

Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of ABC transporterpolypeptide dimers, trimers, tetramers or higher order structures, orstructures formed by ABC transporter polypeptide bound to anotherpolypeptide. ABC transporter polypeptide can be labeled with both adonor and acceptor fluorophore. Upon mixing of the two labeled speciesand excitation of the donor fluorophore, fluorescence energy transfercan be detected by observing fluorescence of the acceptor. Compoundsthat block dimerization will inhibit fluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on ABC transporter polypeptide self-association as well as anassociation of ABC transporter polypeptide and another polypeptide orsmall molecule. ABC transporter polypeptide can be coupled to a sensorchip at low site density such that covalently bound molecules will bemonomeric. Solution protein can then passed over the ABC transporterpolypeptide-coated surface and specific binding can be detected inreal-time by monitoring the change in resonance angle caused by a changein local refractive index. This technique can be used to characterizethe effect of small molecules on kinetic rates and equilibrium bindingconstants for ABC transporter polypeptide self-association as well as anassociation of ABC transporter polypeptide and another polypeptide orsmall molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of ABC transporter polypeptide withanother ABC transporter polypeptide or a different polypeptide. ABCtransporter polypeptide can be coupled to a scintillation-filled bead.Addition of radio-labeled ABC transporter polypeptide results in bindingwhere the radioactive source molecule is in close proximity to thescintillation fluid. Thus, signal is emitted upon ABC transporterpolypeptide binding and compounds that prevent ABC transporterpolypeptide self-association or an association of ABC transporterpolypeptide and another polypeptide or small molecule will diminishsignal.

ICS biosensors have been described by AMBRI (Australian MembraneBiotechnology Research Institute). They couple the self-association ofmacromolecules to the closing of gramacidin-facilitated ion channels insuspended membrane bilayers and hence to a measurable change in theadmittance (similar to impedance) of the biosensor. This approach islinear over six decades of admittance change and is ideally suited forlarge scale, high through-put screening of small molecule combinatoriallibraries.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or othervise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedumder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for ABC transporter agolnists is acompetitive assay that combines ABC transporter and a potential agonistwith ABC transporter-binding molecules, recombinant ABC transporterbinding molecules, natural substrates or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. ABC transporter can be labeled, such as by radioactivity or acolorimetric compound, such that the number of ABC transporter moleculesbound to a binding molecule or converted to product can be deteninnedaccurately to assess the effectiveness of the potential antagonist.

Potential antagonists include, among others, small organic molecules,pcptides, polypeptides and antibodies that bind to a polynucleotideand/or polypeptide of the invention and thereby inhibit or extinguishits activity or expression. Potential antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds the same sites on a binding molecule,such as a binding molecule, without inducing ABC transporter-inducedactivities, thereby preventing the action or expression of ABCtransporter polypeptides and/or polynucleotides by excluding ABCtransporter polypeptides and/or polynucleotides from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56:560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of ABC transporter.

Other examples of potential polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

Certain of the polypeptides of the invention are biomimetics, functionalmimctics of the natural ABC transporter polypeptide. These functionalmimetics may be used for, among other things, antagonizing the activityof ABC transporter polypeptide or as a antigen or iminunogcn in a mannerdescribed elsewhere herein. Functional mimetics of the polypeptides ofthe invention include but are not limited to truncated polypeptides. Forexample, preferred functional mimetics include, a polypeptide comprisingthe polypeptide sequence set forth in SEQ ID NO:2 lacking 20, 30, 40,50, 60, 70 or 80 amino- or carboxy-termninal amino acid residues,including fusion proteins comprising one or more of these truncatedsequences. Polynucleotides encoding each of these functional imineticsmay be used as expression cassettes to express each mimetic polypeptide.It is preferred that these cassettes comprise 5′ and 3′ restrictionsites to allow for a convenient means to ligate the cassettes togetherwhen desired. It is further preferred that these cassettes comprise geneexpression signals known in the art or described elsewhere herein.

Thus, in another aspect, the present invention relates to a screeningkit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for a polypeptide and/or polynucleotide of thepresent invention: or compounds which decrease or enhance the productionof such polypeptides and/or polynucleotides, which comprises: (a) apolypeptide and/or a polynucleotide of the present invention; (b) arecombinant cell expressing a polypeptide and/or polynucleotide of thepresent invention; (c) a cell membrane expressing a polypeptide and/orpolynucleotide of the present invention; or (d) antibody to apolypeptide and/or polynucleotide of the present invention; whichpolypeptide is preferably that of SEQ ID NO:2, and which polynucleotideis preferably that of SEQ ID NO:1.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist, antagonist orinhibitor of the polypeptide and/or polynucleotide, by: (a) detenniningin the first instance the three-dimensional structure of the polypeptideand/or polyiniucleotide, or complexes thereof, (b) deducing thethree-dimensional structure for the likely reactive site(s), bindingsite(s) or motif(s) of an agonist, antagonist or inhibitor; (c)synthesizing candidate compounds that are predicted to bind to or reactwith the deduced binding site(s), reactive site(s), and/or motif(s); and(d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors. It will be further appreciated that this willnormally be an iterative process, and this iterative process may beperfonned using automated and computer-conitrolled steps.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of ABC transporter polypeptide and/or polynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by unhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzynes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the ABCtransporter polypeptidc and/or polypeptide.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG 1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. W094/29458 and W094/22914.

In still another approach, expression of the gene encoding endogenousABC transporter polypeptide can be inhibited using expression blockingtechniques. This blocking may be targeted against any step in geneexpression, but is preferably targeted against transcription and/ortranslation. An examples of a known technique of this sort involve theuse of antisense sequences, either internally generated or separatelyadministered (see, for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al, Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial dnigs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgamo orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial ABC transporter proteins that mediate tissue damage and/or; toblock the normal progression of pathogenesis in infections initiatedother than by the implantation of in-dwelling devices or by othersurgical techniques.

In accordance with yet another aspect of the invention, there areprovided ABC transporter agonists and antagonists, preferablybacteristatic or bactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein “H. pylori”) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofABC transporter polypeptides and/or polynucleotides) found using screensprovided by the invention, or known in the art, particularlynarrow-spectrum antibiotics, should be usefuil in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also prevent, inhibit and/or cure gastric ulcers andgastritis.

Vaccines

There are provided by the invention, products, compositions and methodsfor assessing ABC transporter expression, treating disease, assayinggenetic variation, and administering a ABC transporter polypeptideand/or polyiiucleotide to an organism to raise an immnunologicalresponse against a bacteria, especially a Staphylococcus aureusbacteria.

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with ABC transporter polviucleotideand/or polypeptide, or a fragment or variant thereof, adequate toproduce antibody and/or T cell immune response to protect saidindividual from infection, particularly bacterial infection and mostparticularly Staphylococcus aureus infection. Also provided are methodswhereby such immnunological response slows bacterial replication. Yetanother aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector, sequence or ribozyme to directexpression of ABC transporter polynucleotide and/or polypeptide, or afragment or a variant thereof, for expressing ABC transporterpolynucleotide and/or polypeptide, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual, preferably a human, from disease, whether that disease isalready established within the individual or not. One example ofadmninistering the gene is by accelerating it into the desired cells asa coating on particles or otherwise. Such nucleic acid vector maycomprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNAhybrid, a DNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a ABC transporterpolynucleotide and/or polypeptide encoded therefrom, wherein thecomposition comprises a recombinant ABC transporter polynucleotideand/or polypeptide encoded therefrom and/or comprises DNA and/or RNAwhich encodes and expresses an antigen of said ABC transporterpolynucleotide, polypeptide encoded therefrom, or other polypeptide ofthe invention. The immunological response may be used therapeutically orprophylactically and may take the fonn of antibody immunity and/orcellular inmmunity, such as cellular immunity arising from CTL or CD4+Tcells.

A ABC transporter polypeptide or a fragment thereof may be fused withco-protein or chemical moiety which may or may not by itself produceantibodies, but which is capable of stabilizing the first protein andproducing a fused or modified protein which will have antigenic and/orimmunogenic properties, and preferably protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Hemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, or any otherrelatively large co-protein which solubilizes the protein andfacilitates production and purification thereof Moreover, the co-proteinmay act as an adjuvant in the sense of providing a generalizedstimulation of the immune system of the organism receiving the protein.The co-protein may be attached to either the amino- or carboxy-terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Staphylococcus aureus. Suchexperiments will be particularly useful for identifying protein cpitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Staphylococcius aureus infection, inmammals, particularly humans.

A polypeptide of the invention may be used as an antigen for vaccinationof a host to produce specific antibodies which protect against invasionof bacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, for example, by mechanical, chemical, thermal orradiation damage or by implantation of indwelling devices, or wounds inthe mucous membranes, such as the mouth, throat, mammary glands, urethraor vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken doral in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Fonnulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine fonnulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain ABCtransporter polypeptides and polynucleotides, it is to be understoodthat this covers fragments of the naturally occurring polypeptides andpolynucleotides, and similar polypeptides and polynucleotides withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant polypeptides orpolynucleotides.

Compositions, Kits and Administration

In a further aspect of the invention there are provided compositionscomprising a ABC transporter polynucleotide and/or a ABC transporterpolypeptide for administration to a cell or to a multicellular organism.

The invention also relates to compositions comprising a polyniucleotideand/or a polypeptides discussed herein or their agonists or antagonists.The polypeptides and polynucleotidcs of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for adinnistration to an individual. Such compositionscomprise, for instance, a media additive or a therapeutically effectiveamount of a polypeptide and/or polynucleotide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof The formulation shouldsuit the mode of admiiistration. The invention further relates todiagnostic and pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of the invention maybe employed alone or in conjtmction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollicents in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the fonmulation; moreusually they will constitute up to about 80% by weight of theformulation.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide and/or polynucleotide, such as the soluble form of apolypeptide and/or polynucleotide of the present invention, agonist orantagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or cxcipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable hiffonnationresource with which to determine their 2- and 3-dimensional structuresas well as to identify further sequences of similar homology. Theseapproaches are most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data in a knownmacromolecular structure prograin or to search a sequence database usingwell known searching tools, such as GCC.

The polynucleotide and polypeptide sequences of the invention areparticularly useful as components in databases useful for searchanalyses as well as in sequence analysis algorithms. As used in thissection entitled “Sequence Databases, Sequences in a Tangible Medium,and Algorithms,” and in claims related to this section, the terms“polynucleotide of the invention” and “polynucleotide sequence of theinvention” mean any detectable chemical or physical characteristic of apolynucleotide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form. For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, called bases, and mass spectrographic data. As used in thissection entitled Databases and Algorithms and in claims related thereto,the terms “polypeptide of the invention” and “polypeptide sequence ofthe invention” mean any detectable chemical or physical characteristicof a polypeptide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form. For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, and mass spectrographic data.

The invention provides a computer readable medium having stored thereonpolypeptide sequences of the invention and/or polynucleotide sequencesof the invention. For example, a computer readable medium is providedcomprising and having stored thereon a member selected from the groupconsisting of. a polynucleotide comprising the sequence of apolynucleotide of the invention; a polypeptidc comprising the sequenceof a polypeptide sequence of the invention; a set of polynucleotidesequences wherein at least one of the sequences comprises the sequenceof a polynucleotide sequence of the invention; a set of polypeptidesequences wherein at least one of the sequences comprises the sequenceof a polypeptide sequence of the invention; a data set representing apolynucleotide sequence comprising the sequence of polynucleotidesequence of the invention; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of apolypeptide sequence of the invention; a polyinucleotide comprising thesequence of a polynucleotide sequence of the invention; a polypeptidecomprising the sequence of a polypeptide sequence of the invention; aset of polynucleotide sequences wherein at least one of the sequencescomprises the sequence of a polynucleotide sequence of the invention; aset of polypeptide sequences wherein at least one of said sequencescomprises the sequence of a polypeptide sequence of the invention; adata set representing a polynucleotide sequence comprising the sequenceof a polynucleotide sequence of the invention; a data set representing apolynucleotide sequence encoding a polypeptide sequence comprising thesequence of a polypeptide sequence of the invention. The computerreadable medium can be any composition of matter used to storeinformation or data, including, for example, commercially availablefloppy disks, tapes, chips, hard drives, compact disks, and video disks.

Also provided by the invention are methods for the analysis of charactersequences or strings, particularly genetic sequences or encoded geneticsequences. Preferred methods of sequence analysis include, for example,methods of sequence homology analysis, such as identity and similarityanalysis, RNA stricture analysis, sequence assembly, cladistic analysis,sequence motif analysis, open reading frame determination, nucleic acidbase calling, nucleic acid base trimming, and sequencing chromatogrampeak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of providing a firstpolynucleotide sequence comprising the sequence a polynucleotide of theinvention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing a firstpolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said firstpolypeptide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

A computer based method is still further provided for polynucleotideassembly, said method comprising the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and atleast one second polynucleotide or polypeptide sequence.

A computer based method is still further provided for polynucleotideassembly, said method comprising the steps of: providing a firstpolypeptide sequence comprising a polypeptide of the invention in acomputer readable medium; and screening for at least one overlappingregion between said first polypeptide sequence and at least one secondpolynucleotide or polypeptide sequence.

In another preferred embodiment of the invention there is provided acomputer readable medium having stored thereon a member selected fromthe group consisting of: a polynucleotide comprising the sequence of SEQID NO:1; a polypeptide comprising the sequence of SEQ ID NO:2; a set ofpolynucleotide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO:1; a set of polypeptide sequenceswherein at least one of said sequences comprises the sequence of SEQ IDNO:2; a data set representing a polynucleotide sequence comprising thesequence of SEQ ID NO:1; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of SEQID NO:2; a polynucleotide comprising the sequence of SEQ ID NO:1; apolypeptide comprising the sequence of SEQ ID NO:2; a set ofpolynucleotide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO:1; a set of polypeptide sequenceswherein at least one of said sequences comprises the sequence of SEQ IDNO:2; a data set representing a polynucleotide sequence comprising thesequence of SEQ ID NO:1; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of SEQID NO:2. A further preferred embodiment of the invention provides acomputer based method for perfoniing homology identification, saidmethod comprising the steps of providing a polynucleotide sequencecomprising the sequence of SEQ ID NO:1 in a computer readable medium;and comparing said polynucleotide sequence to at least onepolyiucleotide or polypeptide sequence to identify homology.

A still further preferred embodiment of the invention provides acomputer based method for performing homology identification, saidmethod comprising the steps of: providing a polypcptide sequencecomprising the sequence of SEQ ID NO:2 in a computer readable medium;and comparing said polypeptide sequence to at least one polynucleotideor polypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of SEQID NO:1 in a computer readable medium; and screening for at least oneoverlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polynucleotide sequence comprising the sequence of SEQID NO:1 in a computer readable medium; and comparing said polynucleotidesequence to at least one polynuclcotide or polypeptide sequence toidentify homology.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Antibody(ies)” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Antigenically equivalent derivative(s)” as used herein encompasses apolypeptide, polynucleotide, or the equivalent of either which will bespecifically recognized by certain antibodies which, when raised to theprotein, polypeptide or polynucleotide according to the invention,interferes with the immediate physical interaction between pathogen andmammalian host.

“Bispecific antibody(ies)” means an antibody comprising at least twoantigen binding domains, each domain directed against a differentepitope.

“Bodily material(s)” means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, disease, such as, infections of theupper respiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophtlialmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

“Fusion protein(s)” refers to a protein encoded by two, often unrelated,fused genes or fragments thereof. In one example, EP-A-0464 disclosesfusion proteins comprising various portions of constant region ofimmununoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

“Host cell(s)” is a cell which has been transformed or transfected, oris capable of transformation or transfection by an exogenouspolynucleotidc sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humania Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesincilide, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferrcd meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n_(n)≦x_(n)−(x_(n)•y),

wherein n_(n) is the number of nuclcotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and Wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynuclcotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotidc sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:1, that is itmay be 100% identical, or it may include up to a certain integer numberof nucleic acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is detennined by multiplyingthe total number of nucleic acids in SEQ ID NO:1 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of nucleic acids in SEQ ID NO:1, or:

n_(n)≦x_(n)−(x_(n)•y),

wherein n_(n) is the number of nucleic acid alterations, x_(n) is thetotal number of nucleic acids in SEQ ID NO:1, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO:2 or may include up to a certain integer number ofamino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

 n_(a)≦x_(a)−(x_(a)•y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n_(a)≦x_(a)−(x_(a)•y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

“Immunologically equivalent derivative(s)” as used herein encompasses apolypeptide, polyiucleotide, or the equivalent of either which when usedin a suitable formulation to raise antibodies in a vertebrate, theantibodies act to interfere with the immediate physical interactionbetween pathogen and mammalian host.

“Immunospecific” means that characteristic of an antibody whereby itpossesses substantially greater affinity for the polypeptides of theinvention or the polynucleotides of the invention than its affinity forother related polypeptides or polynucleotides respcctively, particularlythose polypeptides and polnucleotides in the prior art.

“Individual(s)” means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal enviromnent, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the tenn is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Organism(s)” means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Frysipelothrix, Branhamella,Actinohacillus, Streptohacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Frwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G iStreptococcus, Streptococcus pneumoniae, Streptococcuspyogenes, Streptococcus agalactiae, Streptococcus faecalis,Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae,Neisseria meningiticlis, Staphylococcus aureus, Staphylococcusepidermidis, Corynebacterium diptheriae, Gardnerella vaginalis,Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans,Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes,Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica,Escherichia coli, Shigella dysenteriae, Haemophilus influenzae,Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi,Bordetella, Salmonella typhi, Citrobacter frezindii, Proteus mirabilis,Proteus vilgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratiamarcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenteri,Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis,Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridiumperfringens, Clostridium tetani, Clostridium botulinurn, Treponemapallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) anarchaeon, incluhding but not limited to Archaebacter, and (iii) aunicellular or filamentous eukaryote, including but not limited to, aprotozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces,or Candida, and a member of the species Saccharomyces ceriviseae,Kluveromyces lactis, or Candida albicans.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limnitation, single-and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve oinly a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosinc, or modified bases, suchas tritylated bases, to name just tvo examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefuilpurposes knotai to those of skill in the art. The term“polynuclcotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short poly-nucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechnques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl temini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystcine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamla-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS-STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Subtraction set” is one or more, but preferably less than 100,polynucleotides comprising at least one polynucleotide of the invention.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and lIe; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gin; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereothervise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain Selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 ABC Transporter Characterization

The superfamily of ABC transporters transport proteins, peptides,polysaccharides heme, and ions across a cell membrane (Michael J Fathand Roberto Kolter, 1993. Microbial Reviews:57:995-1017). Three majorsubfamilies of ABC transporters have been described. One subfamily, iscomposed of periplasmic perineases. This subfamily features multisubunitimport systems, with a hydrophilic, membrane associated protein carryingthe ABC ATP-binding domain, and a periplasmic binding protein. Theseimporter have the membrane-spanning domain (MSD) and ATP-binding domainon separate subunits.

Bacterial ABC exporters tend to have have the membrane-spanning domain(MSD) and ATP-binding domain on the same subunit. Some bacterialexporters lack MSD. Since gram positive bacteria have a single cellularmembrane, they lack accessory factors used by gram negative bacteria toexport products across the outer membrane.

4 1 702 DNA Staphylococcus aureus CDS (1)...(699) 1 tgt gga tcc tat cgaagc aat tgg agg tgc aga ata atg gca tta gtc 48 Cys Gly Ser Tyr Arg SerAsn Trp Arg Cys Arg Ile Met Ala Leu Val 1 5 10 15 gtt aaa gat atc gtcaaa aat ttc gga gaa ggt ttg tct gaa aca aaa 96 Val Lys Asp Ile Val LysAsn Phe Gly Glu Gly Leu Ser Glu Thr Lys 20 25 30 gtt tta aaa ggt att aatttt gaa gtg gaa caa ggg gaa ttt gtc att 144 Val Leu Lys Gly Ile Asn PheGlu Val Glu Gln Gly Glu Phe Val Ile 35 40 45 tta aat ggt gcc tct ggt tctggg aaa aca aca ttg cta acg ata tta 192 Leu Asn Gly Ala Ser Gly Ser GlyLys Thr Thr Leu Leu Thr Ile Leu 50 55 60 ggc gga ttg tta agt caa acg agtggt aca gtg ctt tac aat gat gca 240 Gly Gly Leu Leu Ser Gln Thr Ser GlyThr Val Leu Tyr Asn Asp Ala 65 70 75 80 cca ttg ttt gat aaa cag cat cctcct agt gat ttg cga ttg gaa gat 288 Pro Leu Phe Asp Lys Gln His Pro ProSer Asp Leu Arg Leu Glu Asp 85 90 95 att ggt att att att caa tct tca catata gtt cct tat tta aaa gtg 336 Ile Gly Ile Ile Ile Gln Ser Ser His IleVal Pro Tyr Leu Lys Val 100 105 110 ata gag caa ttg aca cta gta ggc caagaa gcg gga atg acc aaa caa 384 Ile Glu Gln Leu Thr Leu Val Gly Gln GluAla Gly Met Thr Lys Gln 115 120 125 caa agt tca aca aga gca ata caa cttttg aaa aat att ggg tta gaa 432 Gln Ser Ser Thr Arg Ala Ile Gln Leu LeuLys Asn Ile Gly Leu Glu 130 135 140 gat cgc ttg aat gta tat ccg cat cagtta tct ggc ggt gaa aag caa 480 Asp Arg Leu Asn Val Tyr Pro His Gln LeuSer Gly Gly Glu Lys Gln 145 150 155 160 cgt gtt gcg att atg aga gca tttatg aat aat ccg aaa atc att tta 528 Arg Val Ala Ile Met Arg Ala Phe MetAsn Asn Pro Lys Ile Ile Leu 165 170 175 gca gat gag ccc aca gca agc ttagat gcc gat aga gca aaa aaa gtt 576 Ala Asp Glu Pro Thr Ala Ser Leu AspAla Asp Arg Ala Lys Lys Val 180 185 190 gtt gag atg ata cgt caa caa attaaa gaa caa caa atg att ggt att 624 Val Glu Met Ile Arg Gln Gln Ile LysGlu Gln Gln Met Ile Gly Ile 195 200 205 atg att aca cac gat cga aga ttattt gaa tat gca gat aga gtg ata 672 Met Ile Thr His Asp Arg Arg Leu PheGlu Tyr Ala Asp Arg Val Ile 210 215 220 gaa tta gaa gat ggc aaa ata actgat tag 702 Glu Leu Glu Asp Gly Lys Ile Thr Asp 225 230 2 233 PRTStaphylococcus aureus 2 Cys Gly Ser Tyr Arg Ser Asn Trp Arg Cys Arg IleMet Ala Leu Val 1 5 10 15 Val Lys Asp Ile Val Lys Asn Phe Gly Glu GlyLeu Ser Glu Thr Lys 20 25 30 Val Leu Lys Gly Ile Asn Phe Glu Val Glu GlnGly Glu Phe Val Ile 35 40 45 Leu Asn Gly Ala Ser Gly Ser Gly Lys Thr ThrLeu Leu Thr Ile Leu 50 55 60 Gly Gly Leu Leu Ser Gln Thr Ser Gly Thr ValLeu Tyr Asn Asp Ala 65 70 75 80 Pro Leu Phe Asp Lys Gln His Pro Pro SerAsp Leu Arg Leu Glu Asp 85 90 95 Ile Gly Ile Ile Ile Gln Ser Ser His IleVal Pro Tyr Leu Lys Val 100 105 110 Ile Glu Gln Leu Thr Leu Val Gly GlnGlu Ala Gly Met Thr Lys Gln 115 120 125 Gln Ser Ser Thr Arg Ala Ile GlnLeu Leu Lys Asn Ile Gly Leu Glu 130 135 140 Asp Arg Leu Asn Val Tyr ProHis Gln Leu Ser Gly Gly Glu Lys Gln 145 150 155 160 Arg Val Ala Ile MetArg Ala Phe Met Asn Asn Pro Lys Ile Ile Leu 165 170 175 Ala Asp Glu ProThr Ala Ser Leu Asp Ala Asp Arg Ala Lys Lys Val 180 185 190 Val Glu MetIle Arg Gln Gln Ile Lys Glu Gln Gln Met Ile Gly Ile 195 200 205 Met IleThr His Asp Arg Arg Leu Phe Glu Tyr Ala Asp Arg Val Ile 210 215 220 GluLeu Glu Asp Gly Lys Ile Thr Asp 225 230 3 21 DNA Staphylococcus aureus 3actagtaggc caagaagcgg g 21 4 22 DNA Staphylococcus aureus 4 tgctctcataatcgcaacac gt 22

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide sequence or the full complement of the entire length ofthe polynucleotide sequence, wherein the polynucleotide sequencecomprises SEQ ID NO:1.
 2. A vector comprising the isolatedpolynucleotide of claim
 1. 3. An isolated host cell comprising thevector of claim
 2. 4. A process for producing a polypeptide comprisingculturing the host cell of claim 3 under conditions sufficient for theproduction of the polypeptide, wherein the polypeptide comprises SEQ IDNO:2.
 5. The isolated polynucleotide of claim 1 encoding a fusionpolypeptide, wherein the fusion polypeptide comprises SEQ ID NO:2.
 6. Anisolated polynucleotide comprising a polynucleotide sequence, whereinthe polynucleotide sequence encodes a polypeptide comprising SEQ IDNO:2.
 7. A vector comprising the isolated polynucleotide of claim
 6. 8.An isolated host cell comprising the vector of claim
 7. 9. A process forproducing a polypeptide comprising culturing the host cell of claim 8under conditions sufficient for the production of the polypeptide,wherein the polypeptide comprises SEQ ID NO:2.
 10. The isolatedpolynucleotide of claim 6 which encodes a fusion polypeptide; whereinthe fusion polypeptide comprises SEQ ID NO:2.
 11. An isolatedpolynucleotide comprising a polynucleotide sequence, wherein thepolynucleotide sequence encodes a polypeptide consisting of SEQ ID NO:2.12. A vector comprising the isolated polynucleotide of claim
 11. 13. Anisolated host cell comprising the vector of claim
 12. 14. A process forproducing a polypeptide comprising culturing the host cell of claim 13under conditions sufficient for the production of the polypeptide,wherein the polypeptide consists of SEQ ID NO:2.